Sporamin induces apoptosis and inhibits NF-κB activation in human pancreatic cancer cells

Abstract

Sporamin, a Kunitz-type trypsin inhibitor (TI) from sweet potato tuberous roots, has demonstrated anti-tumor activity through poorly defined mechanisms. Furthermore, the effects of sporamin on pancreatic cancer have not been explored. Herein, we studied the effects of sporamin on two human pancreatic cancer cell lines, PANC-1 and BxPC-3. Sporamin significantly inhibited the cell viability and proliferation activity and induced apoptosis in PANC-1 and BxPC-3 cells. Consistently, in sporamin-treated PANC-1 and BxPC-3 cells, the anti-apoptotic proteins Bcl-2 and Bcl-XL were downregulated and the pro-apoptotic protein Bax was upregulated. Moreover, nuclear factor kappa B activation and IκBα phosphorylation were inhibited, and total IκBα expression was increased in sporamin-treated PANC-1 and BxPC-3 cells. Thus, our results suggest that the anti-tumor effects of sporamin in pancreatic cancer cells might result partly from induction of apoptosis by downregulating nuclear factor kappa B pathway.

Introduction

Pancreatic cancer (PC) is one of the most prevalent tumors in the world. ¹ Current treatment protocols for advanced PC often include traditional treatments, such as surgery, chemotherapy, and radiation therapy. ² Despite advances in the traditional treatments of PC, PC mortality has not improved significantly,^1,2 and it is apparent that different approaches to treat PC are needed. Therefore, investigations of potential alternative therapies for PC with fewer associated toxicities are continuing.

Nuclear factor kappa B (NF-κB), an inducible transcription factor, plays a crucial role in pancreatic carcinogenesis.^3,4 In non-stimulated cells, NF-κB resides in the cytoplasm in a complex with the inhibitor protein, collectively called IκBs (Inhibitors of κB).^3,4 Most agents that activate NF-κB use a common pathway based on the phosphorylation of the two NH2-terminal serines in IκBs with subsequent ubiquitination and degradation of IκBs. ⁵ Phosphorylation and degradation of IκBs result in nuclear translocation of released NF-κB dimers (p50/p65) and activation of target genes, such as Bcl-2 and Bcl-XL.^5,6

Chemotherapy-induced tumor cell apoptosis by regulation of signaling pathways has become the principle strategy in tumor chemotherapeutics. ⁷ However, most of the chemotherapeutics for PC have great aversive side effects, ² so it is necessary for us to find a proper therapeutic strategy for PC patients. Sporamin, a Kunitz-type trypsin inhibitor (TI), is the major soluble storage protein in sweet potato tuberous roots, which can induce anti-tumor effects in several cancer cell lines, such as human leukemia HL60 cells, human tongue cancer Tca8113 cells, and human colorectal cancer HCT-8 cells.^8–10 However, the effects of sporamin on PC cells have not been previously explored. Thus, we investigated the effects of sporamin on cell viability, cell proliferation activity, and apoptosis in human PC cell lines PANC-1 and BxPC-3. Furthermore, we explored the effects of sporamin on NF-κB activation and its related genes to provide more insights into the mechanism behind the anti-tumor effects of sporamin on PC cells.

Materials and methods

Materials

Sporamin was purified from fresh sweet potato tuberous roots, as reported previously. ⁹ Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (Carlsbad, CA, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), [³H] thymidine, and dimethyl sulfoxide (DMSO) were bought from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

PANC-1 and BxPC-3 (American Type Culture Collection (ATCC), Manassas, VA, USA), two human PC cell lines, were cultured in DMEM with 10% heat-inactivated FBS, benzylpenicillin (100 kU/L), and streptomycin (100 mg/L) at 37°C in an incubator containing humidified air with 5% CO₂.

[³H] thymidine incorporation assay

PANC-1 and BxPC-3 cells were plated on 24-well plates and grown to 80% confluency in DMEM with 10% dialyzed and charcoal-stripped FBS. The cell cultures were then rinsed in phenol-free DMEM medium and incubated with respective test substances in phenol-free and serum-free DMEM for 48 h. [³H] thymidine (1.35 × 10⁴ Bq/L) was added. Cells were cultured for another 12 h. The supernatant was aspirated and then washed twice with phosphate-buffered saline (PBS) to remove excess [³H] thymidine. The cells were then dissolved in 0.2 mol/L of NaOH. Radioactivity (incorporation activity) was determined by scintillation counter.

Cell viability assay

PANC-1 and BxPC-3 cells were plated on 96-well plates and grown to 80% confluency. The cell cultures were then rinsed in phenol-free DMEM medium and incubated with respective test substances in phenol-free and serum-free DMEM for 48 h. At the end of this time interval, MTT was added to a final concentration of 0.5 mg/mL. After 1 h of incubation, cultures were removed from the incubator and the formazan crystals were solubilized by adding solubilization solution including 10% (v/v) Triton X-100 and 0.1 mol/L HCl in isopropanol equal to the volume of original culture medium. Colorimetric determination of MTT reduction was made at 570 nm using a microplate enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad, Hercules, CA, USA).

Morphological determination and quantification of apoptosis

PANC-1 and BxPC-3 cells were cultured on 22 mm × 22 mm glass coverslips on six-well plates at a density of 2 × 10⁵ per well and incubated with or without indicated treatments for the desired time. For nuclear staining assay, cells were fixed with 4% cold paraformaldehyde (pH 7.4) and stained with 0.5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA) for 5 min at room temperature. The coverslips were rinsed with PBS and mounted onto slides with a fluorescent mounting medium. The cells were then visualized under a fluorescence microscope (DMI4000B; Olympus, Tokyo, Japan).

Flow cytometry analysis for apoptosis

Apoptosis were measured using the Annexin V-PE/7-AAD Apoptosis Detection Kit (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, the cells were incubated with or without indicated treatments for the desired time. Attached and floating cells were pooled, pelleted by centrifugation, washed two times with cold PBS, and then resuspended in 1× binding buffer (10 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES), pH 7.4, 140 mM NaOH, and 2.5 mM CaCl₂) at a concentration of 1 × 10⁶ cells/mL. A volume of 1 × 10⁵ cells were transferred to a 5-mL tube and stained with 5 µL of Annexin V-PE and 5 µL of 7-amino-actinomycin D (7-AAD) for 15 min at room temperature in the dark. Finally, 400 µL of 1× binding buffer was added to each tube and apoptosis was determined using a flow cytometer (BD, Franklin Lakes, NJ, USA).

Western blotting

Cellular total, nuclear, and cytoplasmic protein extractions were carried out using M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) and Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturers’ instructions. Cell protein extracts were collected and centrifuged at 14,000g in a microcentrifuge for 10 min at 4°C. The supernatants were collected, and the protein concentrations were determined using Bradford’s method. Cell protein extracts were either used immediately or stored at −70°C.

For polyacrylamide gel electrophoresis (PAGE), 20 µg of proteins from each cell extract was loaded into each well of the gel and electrophoresed at 98 V. Proteins on the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane by conventional tank-buffer electrotransfer for 2 h. The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.05% Tween 20 for 1 h. The primary antibodies (all used at a 1:200 dilution) against Bcl-2, Bcl-XL, Bax, IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphor-IκBα (serine 32; Cell Signaling Technology, Danvers, MA, USA), tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) were added separately and incubated with the membrane for overnight at 4°C before the membrane was washed three times with TBS/Tween 20. Secondary antibody conjugated to horseradish peroxidase at a 1:2000 dilution was added to the membrane and was incubated at room temperature with gentle agitation. After 1 h, the membrane was washed with four changes of TBS/Tween 20, at 5 min per wash. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) Kit (Pierce) and scanned using an LAS-4000 imaging system (Fujifilm, Tokyo, Japan).

Cellular immunofluorescence

Cells were cultured on 22 mm × 22 mm glass coverslips on six-well plates at a density of 2 × 10⁵ per well, allowed to adhere overnight, and treated with indicated concentrations of sporamin or their control vehicles. After 48 h, the cells were washed twice with cold PBS, fixed in 4% cold paraformaldehyde (pH7.4) for 15 min, permeabilized in 0.2% Triton X-100 in double-distilled water for 10 min, blocked with 10% normal non-immune goat serum/5% BSA/PBS at 37°C for 30 min, and then incubated overnight at 4°C with different primary antibody in 5% BSA/PBS solution. Subsequently, fluorescent secondary antibody was added at 37°C for 1 h. Nuclei were counterstained with 0.5 µg/mL DAPI at room temperature for 5 min. The coverslips were rinsed with PBS and mounted onto slides with a fluorescent mounting medium. Images were acquired with the fluorescence microscope (DMI4000B; Olympus).

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed using a gel shift assay system kit (Promega, Madison, WI, USA). The NF-κB oligonucleotide probe (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was end-labeled with [γ-³²P] adenosine triphosphate (ATP; Free Biotech, Beijing, China) with T4-polynucleotide kinase. Nuclear protein (2 µg) was pre-incubated in binding buffer (10 mM Tris-Cl, pH 7.5; 50 mM NaCl; 1 mM MgCl₂; 0.5 mM ethylenediaminetetraacetic acid (EDTA); 4% glycerol; 0.5 mM dithiothreitol (DTT), and 0.05 g/L of poly (deoxyinosinic-deoxycytidylic acid)) for 15 min at room temperature. After addition of the 1 µL ³²P-labeled oligonucleotide probe, the incubation was continued for 30 min at room temperature. The reaction was stopped by adding 1 µL of gel loading buffer, and the mixture was subjected to non-denaturing 4% polyacrylamide gel electrophoresis in 0.5× Tris/borate/EDTA (TBE) buffer. The gel was vacuum-dried and exposed to X-ray film at −70°C. The specificity of binding was also examined by competing with the unlabeled oligonucleotide. For EMSA supershift assays, nuclear protein extracts prepared from sporamin-treated cells were incubated with antibodies against either NF-κB p50 or NF-κB p65 for 30 min at room temperature before the complex was analyzed by EMSA. Antibodies against pre-immune serum were included as negative controls. The dried gels were visualized by autoradiography using X-ray film.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Significance of differences between two groups was determined by Scheffé’s modified t test. A p value <0.05 represented a statistically significant difference.

Results

Sporamin inhibits cell viability of PC cells

We tested the ability of sporamin to inhibit the cell viability of two PC cell lines, PANC-1 and BxPC-3, via the MTT assay (Figure 1). PANC-1 and BxPC-3 cells were treated with sporamin at increasing concentrations (0, 12.5, 25, 50, and 100 µM) and for varying times (24, 48, and 72 h). Sporamin was found to inhibit cell viability in a concentration-dependent and time-dependent manner as compared to diluent control.

Figure 1.

Concentration-dependent and time-dependent effects of sporamin-induced cell viability inhibition of PC cells. PANC-1 and BxPC-3 cells were incubated in the absence or presence of the indicated concentrations of sporamin for different incubation times. Then, the cell viability was determined using the MTT assay (points: mean of four determinations (each in quadruplication)); data are shown as mean ± SD (n = 4 experiments; ^Δp < 0.05; ^ΔΔp < 0.01 vs Control (for the comparison of different time points); *p < 0.05; **p < 0.01 vs Control (for the comparison of different concentration points)).

A more significant decrease in cell viability in a concentration-dependent manner of the PC cells occurred after exposure for 48 h to sporamin compared with the diluent-exposed control cells. Therefore, we treated the PC cells with varying concentrations of sporamin for 48 h for further studies.

Sporamin inhibits cell proliferation activity of PC cells

To test the effects of sporamin on the cell proliferation activity of PANC-1 and BxPC-3 cells, [³H] thymidine incorporation assay was used. As shown in Figure 2, after treatment with sporamin at 25, 50, and 100 µM for 48 h, the [³H] thymidine incorporation activity (Bq/min) of PANC-1 and BxPC-3 cells decreased in a concentration-dependent manner as compared to diluent control.

Figure 2.

Effects of sporamin on [³H] thymidine incorporation activity of PC cells. PANC-1 and BxPC-3 cells were treated with sporamin for 48 h and their incorporation activity was determined. Data are shown as mean ± SD (n = 4 experiments; *p < 0.05; **p < 0.01 vs Control).

Sporamin induces cell apoptosis in PC cells

To test whether sporamin induces apoptosis in PC cells, sporamin-treated PANC-1 and BxPC-3 cells were examined under fluorescence microscopy after DAPI nuclear staining. The exposure to sporamin (50 µM, 48 h) resulted in a considerable number of PANC-1 and BxPC-3 cells exhibiting condensed chromatin and fragmented nuclei, the typical markers of apoptosis (Figure 3(a)). After sporamin treatment of 25, 50, or 100 µM for 48 h, the number of apoptotic cells (proportion of apoptotic cells (% of total cells)) was increased significantly as compared to diluent control (Figure 3(b)).

Figure 3.

(a) Effects of sporamin on apoptosis of PC cells. PANC-1 and BxPC-3 cells were treated with or without 50 µM sporamin for 48 h, and then stained with DAPI and photographed under fluorescence microscopy. Condensed and fragmented nuclei appeared in sporamin-treated cells (magnification: 400×). Apoptotic morphological changes in the nucleus were easily distinguishable from intact nuclei and counted, and the percentages were subsequently calculated. Six randomly chosen fields of view were observed with a minimum number of 500 cells scored in each condition. (b) The percentage of apoptotic cells was calculated with total PC cells. (c) The cells were stained with Annexin V-PE/7-AAD and analyzed by flow cytometry (lower left quadrants: viable cells; lower right quadrants: early apoptotic cells (Annexin V+, 7-AAD−); upper right quadrants: non-viable, late apoptotic/necrotic cells (Annexin V+ and 7-AAD+)). The numerical results represent the mean of triplicate plates, and a representative experiment is shown. (d) Bar graphs demonstrated the percentage of apoptotic rates. Data are shown as mean ± SD (n = 4 experiments; *p < 0.05; **p < 0.01 vs Control).

Since sporamin induced marked morphological changes of apoptosis at the concentration of 50 µM for 48 h, PANC-1 and BxPC-3 cells were treated with sporamin (50 µM, 48 h) and stained with Annexin V and 7-AAD to ananlyze apoptosis using flow cytometry. The percentages of apoptotic cells (Annexin V positive) were increased significantly after sporamin treatment (50 µM, 48 h) compared to the control diluent (Figure 3(c) and (d)). These results demonstrate that sporamin can induce cell apoptosis in PC cells.

Sporamin induces downregulation of Bcl-2 and Bcl-XL and upregulation of Bax in PC cells

To address whether Bcl-2, Bcl-XL, and Bax protein expression levels correlate with the apoptosis that occurs after treatment of PANC-1 and BxPC-3 cells with sporamin, the amounts of these proteins in total cell extracts were quantified by western blotting. After sporamin treatment of 25, 50, or 100 µM for 48 h, the Bcl-2 and Bcl-XL expression decreased with evident increases in Bax expression in PANC-1 and BxPC-3 cells (Figure 4). These results indicated that sporamin induced a reduction in heterodimerization of Bcl-2/Bcl-XL with Bax.

Figure 4.

Effects of sporamin treatment on the expression of Bcl-2, Bcl-XL, and Bax. The protein expression of Bcl-2, Bcl-XL, and Bax in PANC-1 and BxPC-3 cells was detected by western blotting using lysates from PANC-1 and BxPC-3 cells treated with varying concentrations of sporamin for 48 h. Each of the blots shown was demonstrated to have equal protein loading by reprobing with the antibody for GAPDH. GAPDH was used as a loading control for the whole cell extracts. Data are representative of at least two independent experiments with similar results.

Sporamin inhibits constitutive NF-κB activation in PC cells

Because both Bcl-2 and Bcl-XL can be regulated by NF-κB in some cells,^6,11 we postulated that sporamin might downregulate Bcl-2 and Bcl-XL expression by inhibiting NF-κB activation. To investigate the effects of sporamin on constitutive NF-κB activation in PC cells, we prepared the nuclear extracts of PANC-1 and BxPC-3 cells treated with various concentrations of sporamin or control diluent and examined constitutive NF-κB activation by EMSA. As shown in Figure 5(a), a significant decrease of NF-κB activation was observed in PANC-1 and BxPC-3 cells treated with varying concentrations of sporamin (0, 25, 50, and 100 µM).

Figure 5.

Effects of sporamin on constitutive NF-κB activation in PANC-1 and BxPC-3 cells. (a) Sporamin inhibits constitutive NF-κB activation in a concentration-dependent manner in PANC-1 and BxPC-3 cells. (b) Nuclear extracts from PANC-1 and BxPC-3 cells incubated with 0–100 µM sporamin for 48 h were subjected to EMSA to assess NF-κB DNA-binding activity. The radioactive bands were quantitated using a PhosphorImager using ImageQuant software. The levels of NF-κB DNA-binding activity relative to control are indicated at the top of each band. Supershift assay of 50 µM sporamin-treated PANC-1 and BxPC-3 cells with specific NF-κB antibody. Data are representative of at least two independent experiments with similar results. The NF-κB-specific complex and the free DNA probe are indicated (arrows). (c) NF-κB p50 and NF-κB p65 expression were measured in PANC-1 cells by immunofluorescence. Representative fluorescence micrograph images of NF-κB p50 and NF-κB p65 (red), nuclei (DAPI: blue), and Tubulin (green).

To further confirm the specificity of NF-κB DNA binding, we performed an EMSA supershift assay with antibodies specific for NF-κB p65 and NF-κB p50. For supershift assays, nuclear protein extracts prepared from sporamin (50 µM, 48 h)-treated cells were incubated with antibodies against either NF-κB p50 or NF-κB p65 for 30 min at room temperature before the complex was analyzed by EMSA. As shown in Figure 5(b), a strong supershift in the case of anti-NF-κB p65, but a weaker shift for anti-NF-κB p50 to a higher molecular weight band, suggesting that the observed NF-κB band consisted of these two subunits, NF-κB p50 and NF-κB p65, and sporamin inhibited NF-κB p65 and NF-κB p50 DNA-binding activity in PANC-1 and BxPC-3 cells. Interestingly, we found that sporamin decreased the nuclear expression of NF-κB p50 and NF-κB p65 in PANC-1 cells (Figure 5(c)). These results indicated that sporamin inhibited NF-κB activation.

Sporamin inhibits IκBα phosphorylation and increases total IκBα expression in PC cells

We examined whether the inhibition of NF-κB activation by sporamin was due to decreased degradation of IκBα. As shown in Figure 6, when PANC-1 and BxPC-3 cells were treated with sporamin with varying concentrations (25, 50, or 100 µM), significant concentration-dependent increases in total IκBα expression levels in cytoplasmic fractions were observed in PANC-1 and BxPC-3 cells (Figure 6).

Figure 6.

Effects of sporamin on the phosphorylation and degradation of IκBα in PC cells. The cytosolic extracts were isolated and subjected to western blot analysis with anti-IκBα antibody. The whole cell extracts were isolated and subjected to western blot analysis using anti-p-IκBα (serine 32) antibody. Each of the blots shown was demonstrated to have equal protein loading by reprobing with the antibody for tubulin or GAPDH. Tubulin and GAPDH were used as loading controls for the cytoplasmic fractions and the whole cell extracts, respectively. Data are representative of at least two independent experiments with similar results.

Because degradation of IκBα normally requires the inhibitor to be phosphorylated, ⁵ it was of interest to examine the extent of IκBα phosphorylation in sporamin-treated PANC-1 and BxPC-3 cells as compared to diluent control. When PANC-1 and BxPC-3 cells were treated with sporamin with varying concentrations (25, 50, or 100 µM) followed by western blotting for the phosphorylated form of IκBα (p-IκBα), concentration-dependent decreases in expression of p-IκBα were observed in total cell extracts of PANC-1 and BxPC-3 cells (Figure 6). These results indicated that sporamin suppressed NF-κB activity might be partly through dephosphorylation of IκBα.

Discussion

Our results in this study demonstrate that sporamin is a strong suppressor of cell growth by inhibiting cell viability and proliferation activity in PANC-1 and BxPC-3 cells. We also found that sporamin exerts the cell growth–suppressive effects by inducing apoptosis in PANC-1 and BxPC-3 cells. Moreover, the sporamin-induced cell growth–suppressive effects in PANC-1 and BxPC-3 cells may be mediated, at least in part, by inhibition of NF-κB activation.

Apoptosis is an important mechanism to eliminate unwanted cells in a wide variety of physiological processes, and deregulation of this process is implicated in pancreatic carcinogenesis.^12,13 Inhibition of cell viability and proliferation activity could be attributable to the induction of cell apoptosis in PC cells.^12,13 Therefore, whether the cell growth suppression observed was induced by apoptosis was then examined. In this study, treatment of PANC-1 and BxPC-3 cells with sporamin showed DNA fragmentation or nuclear condensation in the nucleus measured using DAPI staining, which is typically observed in apoptotic cells, and a significant increase in the percentage of apoptotic cells was confirmed by flow cytometer.

To further elucidate the mechanism of sporamin-induced apoptosis in PC cells, we investigated the important apoptotic regulators, such as Bcl-2, Bcl-XL, and Bax.^11,14–16 Bcl-2, an anti-apoptotic factor, negatively regulates apoptosis, whereas another Bcl-2-homologous protein, Bax, promotes apoptosis by competing with Bcl-2.^11,14–16 Because apoptosis can be regulated by Bcl-2, Bcl-XL, and Bax, we postulated that sporamin might mediate its effects on apoptosis through Bcl-2, Bcl-XL, and Bax. Consistently with the hypothesis, our study showed that sporamin treatment reduces Bcl-2 and Bcl-XL protein levels and increases the pro-apoptotic protein Bax levels. Bcl-2 and related anti-apoptotic proteins seem to dimerize with a pro-apoptotic protein Bax and modulate the sensitivity of cell to apoptosis.^11,14–16 Therefore, our study results might indicate that sporamin induced apoptosis through a reduction in heterodimerization of Bcl-2/Bcl-XL with Bax, then inhibited cell proliferation activity and cell viability.

NF-κB, an important signal transduction pathway, is known to enhance tumor cell growth and inhibit apoptosis through the regulation of growth factors such as Bcl-2 and Bcl-XL.^{17 –19} Since both Bcl-2 and Bcl-XL are NF-κB target genes, which can be regulated by NF-κB,^11,17–19 we here observed that effects of sporamin on the activation of NF-κB, an important transcription factor in the regulation of the genes governing apoptosis. In this study, sporamin could induce downregulation of NF-κB activation, accompanied by decreased expression of Bcl-2 and Bcl-XL, suggesting that sporamin could inhibit expression of Bcl-2 and Bcl-XL, at least in part, through the inhibition of NF-κB activation in PC cells. Our previous experiments showed that sporamin induces apoptosis in tongue cancer cells by downregulation of AKT. ⁹ In addition, NF-κB can be activated through some other signaling pathways, such as AKT signaling. ²⁰ Therefore, sporamin might inhibit NF-κB activation via AKT signaling in PC cells. However, the different effects and mechanisms of sporamin on NF-κB activation may be due to the different tumor cell types with different genetic/epigenetic status. Furthermore, in-depth studies in other tumor cell lines are required.

NF-κB exists as a heterodimer of p50 and p65 subunits and is sequestered in the cytoplasm as an inactive complex bound to an endogenous inhibitor IκB. ²¹ Here, we demonstrated that sporamin mediates an obvious decrease in constitutive NF-κB activation by inhibiting NF-κB p50 and NF-κB p65 DNA-binding activity, representing NF-κB activation, in PC cells. One major mechanism of NF-κB activation is through inhibition of phosphorylation of IκB.^3,4 Following cellular stimulation, IκB proteins are phosphorylated at two specific serine residues at the N-terminus IκBα (serine 32/36) or IκBβ (Ser19/23) by IκB kinase, the phosphorylation of IκB promotes its ubiquitination and degradation, and this would result in the retention of NF-κB in the cytoplasm.^4,21 Degradation of IκB protein in the cytoplasm liberates NF-κB allowing it to translocate into the nucleus.^4,21 In this study, an obvious decrease in constitutive NF-κB activation was paralleled by an obvious increase in cytoplasmic levels of IκBα in PC cells after sporamin treatment.

Consistently, this increase in cytoplasmic levels of IκBα was paralleled by a decrease in cell total protein levels of phosphorylated IκBα. Therefore, our results indicated that sporamin significantly inhibits NF-κB activation by reducing the levels of phosphorylated IκBα, leading to blockage of IκBα proteolytic pathway in NF-κB activation in PC cells. Although our studies here have shown the inhibition of NF-κB DNA-binding activity and phosphorylation of IκBα clearly pointed to NF-κB inhibition in sporamin-treated PC cells. Future applications include the approaches, such as IκBα dephosphorylation or phosphorylation inhibitors, that should be used to explore the exact mechanisms of decreased degradation of IκBα in sporamin-treated PC cells.

In summary, we found that sporamin could inhibit cell viability and proliferation activity and induce apoptosis in PC cells. In addition, we demonstrated that sporamin could lead to NF-κB inhibition in PC cells, accompanied by decreased Bcl-2 and Bcl-XL expression and increased Bax expression. Thus, this supports the effect of sporamin on cell apoptosis regulation. Further identification of additional downstream effectors of sporamin and a better understanding of the underlying mechanisms will help guide the development of more effective agents to treat human PC.